Biologists often need to introduce into living cells a wide range of substances which are normally excluded from the cell by cell walls and outer cell membranes. Such substances include biological stains, proteins, nucleic acids, organelles, chromosomes, and nuclei.
The importance of introducing biological substances into cells is reflected by the large amount of work which has been done in this area, and the expensive technologies which have been developed to achieve this end. While diverse applications of biological delivery systems are known (Introduction of Macro molecules into Viable Mammalian Cells, (Ed. R. Baserga, C. Crose, G. Rovera), Winstar Symposium Series VI, 1980, A. R. Liss Inc., New York), one application of central importance will clearly be the introduction of genetic material into cells for the purpose of genetic engineering. Existing technologies for transporting genetic material into living cells involve uptake mechanisms, fusion mechanisms, and microinjection mechanisms.
Uptake mechanisms include: (a) induction of enhanced membrane permeability by use of Ca.sup.++ and temperature shock (Mandel, M. and Higa, A., 1970, "Calcium Dependent Bacteriophage DNA Infection," J. Mol. Biol., 53: 159-162; Dityatkin, S. Y., Lisovskaya, K. V., Panzhava, N. N., Iliashenko, B. N., 1972, "Frozen-thawed Bacteria as Recipients of Isolated Coliphage DNA", Biochimica et Biophysica Acta, 281: 319-323); (b) use of surface binding agents such as polyethylene glycol (PEG) (Chang, S. and Cohen, S. N., 1979, "High Frequency Transformation of Bacillus subtilis Protoplasts by Plasmid DNA", Mol. Gen. Genet., 168: 111-115; Krens, F. A., Molendijk, L., Wullens, G. J., and Schilperoort, R. A., 1982, "In vitro Transformation of Plant Protoplasts with Ti-Plasmid DNA", Nature, 296: 72), or Ca(PO.sub.4).sub.2 (Graham, F. L., and van der Eb, A. J., 1973, "A New Technique for the Assay of Infectivity of Human Adenovirus 5 DNA", Virology, 52: 456; Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., Lacey, E., Maniatis, T., Silverstein, S., and Axel, R., 1979, "Transformation of Mammalian Cells with Genes from Procaryotes and Eucaryotes", Cell 16: 777): and (c) phagocytosis of particles such as liposomes (Uchimiya, Transfer of Plasmid DNA into Plant Protoplasts", In: Fujiwara A. (ed.), Proc. 5th Intl. Cong. Plant Tissue and Cell Culture, Jap. Assoc for Plant Tissue Culture, Tokyo, pp. 507-508), organelles (Potrykus, I., 1973, "Transplantation of Chloroplasts into Protoplasts of Petunia", Z. Pflanzenphysiol., 70: 364-366) bacteria (Cocking, E. C., 1972, "Plant Cell Protoplasts Isolation and Development", Ann. Rev. Plant Physiol., 23: 29-50), into the cell. These uptake mechanisms generally involve suspensions of single cells, from which any existing cell wall materials have been removed enzymatically.
Uptake protocols are generally quite simple, and allow treatment of large numbers of cells en masse. However, such methods tend to have very low efficiency. In plant protoplasts, transformation frequencies tend to be one in 10,000 or less, while in animal cell uptake systems, transformation frequencies tend to be even lower. In such systems most cells are unaffected, and special cell selection procedures are required to recover the rare cells which have been transformed (Power, J. B. and Cocking, E. C., 1977, "Selection Systems for Somatic Hybrids", In: Reinert, J. and Bajaj, Y.P.S. (eds.) Plant Cell, Tissue, and Organ Culture, Springer-Verlag, N.Y., pp. 497-505).
Fusion mechanisms incorporate new genetic material into a cell by allowing one cell to fuse with another cell. A variation on this procedure involves "ghost" cells. The membrane of killed cells are allowed to fill with a given DNA solution, such that cell fusion incorporates the DNA from the carrier "cell" into the living cell. Cell-to-cell fusion can be induced with the aid of substances such as PEG (Bajaj, Y.P.S., 1982, "Protoplast Isolation, Culture and Somatic Hybridization", In: Reinert, J. and Bajaj, Y.P.S. (eds.) Plant Cell, Tissue, and Organ Culture, Springer-Verlag, N.Y. pp. 467-496), and Sendai virus particles (Uchida, T., Yamaizumi, M., Mekada, E., Okada, Y., 1980, "Methods Using HVJ (Sendai Virus) for Introducing Substances into Mammalian Cells:, In: Introduction of Macro molecules into Viable Mammalian Cells, Windsor Symposium Series V.I. A. R. Liss Inc., N.Y., pp. 169-185).
As with uptake mechanisms, fusion technologies rely upon the use of single cell suspensions, where cells are enzymatically stripped of any cell wall material. While fusion technologies can have relatively good efficiencies in terms of numbers of cells affected the problems of cell selection can be more complex, and the resulting cells are typically of elevated ploidy, which can limit their usefulness.
Microinjection technologies employ extremely fine, drawn out capillary tubes, which are sometimes called micropipettes. These capillary tubes can be made sufficiently small to be used as syringe needles for the direct injection of biological substances into certain types of individual cells (Diacumakos, E. G., 1973, "Methods for Microinjection of Human Somatic Cells in Culture", In: Prescott DM (ed.) Methods in Cell Biology, Academic Press, N.Y. pp. 287-311; Graessmann, M. and Graessman, A., 1983, "Microinjection of Tissue Culture Cells", Methods in Enzymology, 101: 482-492). When small cells need to be injected, very sharp microelectrodes are required, whose tips are very easily broken or clogged. Very high pressures are required to cause bulk flow through orifices smaller than one micron. Regulation of such bulk flow is very difficult. The entire process is something of an art, requiring different modifications for different cell types. One modification of microinjection involves pricking with a solid-glass drawn needle, which carries in biological solutions which are bathing the cell (Yamamoto, F., Furusawa, M., Furusawa, I., and Obinata, M., 1982, "The "Pricking" Method", Exp. Cell Res., 142: 79-84). Another modification, called ionophoresis (Purres, R. D., 1981, Microelectrode Methods for Intracellular Recording and Ionophoresis, Academic Press, N.Y., p. 146; Ocho, M., Nakai, S., Tasaka, K., Watanabe, S., and Oda, T., 1981, "Micro-injection of Nucleic Acids Into Cultured Mammalian Cells by Electrophoresis", Acta Med. Okayama, 35(5): 381-384), uses electrophoresis of substances out of the microelectrode and into the cell, as an alternative to high pressure bulk flow.
Microinjection procedures can give extremely high efficiencies relative to delivery into the cell. Because of this, microinjection has been used successfully in the transformation of individual egg cells. One disadvantage of microinjection is that it requires single cell manipulations and is therefore inappropriate for treating masses of cells and is generally a very tedious and difficult technology. Microinjection is a technology which would not be easily universalized or automated.
In addition to mechanical delivery systems, there exist several infectious agents which can deliver nucleic units into cells. Of primary importance are the well-known retroviral vectors for animal cells, and the Agrobacterium Ti-plasmid vectors for dicot plant cells. Other viruses may also be developed as effective genetic vectors. The problem with infectious agents as DNA delivery systems is two-fold. Firstly, infectious agents have limited host ranges. A given strain of Agrobacterium may only infect a few species of plant, and Agrobacterium as a whole is largely limited to infection of dicot plants (not monocots). Likewise, retroviruses and their promoters tend to be species and tissue specific. Secondly, infectious agents add an additional second living system with all its concominant complications. Thirdly, infectious agents are potentially dangerous--they may harm the organism being modified, or they may lend, through recombination, to the evolution of new pathogens.
While a variety of biological delivery systems presently exist, none of these technologies is free from major limitations. Perhaps the greatest single limitation of all of these technologies is that they are limited to single cell in vitro systems.
It is noted that liquid sprays and liquid jets have been used for inoculation of host plants and animals with virus (and vaccines). The present invention is distinct from these methods.
Devices which are used for rapid human vaccinations have been used for inoculation virus into plants (Mumford, D. L., 1972, Phytopathology, 62:1217-1218). This type of device injects a high-pressure liquid Jet through the epidermis, into the sub-epidermis in a manner similar to a hypodermic needle. Individuals cells are not penetrated, and particles are not involved.
An artist's airbrush can be used to inoculate plants with virus, by spraying a liquid stream containing virus and an abrasive such as carborundum (Pring, D. R. and D. J. Gumpf, 1970, Plant Disease Reporter, 54:550-553). In this case particles are involved, but they are very large particles (&gt;50 micron), and are clearly serving as a general abrasive and not as a vehicle for carrying DNA into cell interiors. The particles themselves are larger than the cells, and could not possibly enter into the cells in a non-lethal manner.
In both of these cases, the virus is being introduced as an infectious entity (complete viral particles), into susceptible host tissue. Comparable inoculations can be made by simply rubbing the virus into the tissue, with or without abrasives. This is clearly very different from the intra-cellular delivery process described and claimed herein, which can introduce naked RNA molecules into cell interiors of non-host tissues.